Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting a wide variety of mammals including sheep and goats (scrapie), cervid spp. (chronic wasting disease), and humans (Creutzfeldt-Jakob disease). Our studies are focused on the prion protein (PrP) due to the critical role of this protein in controlling many aspects of TSE pathogenesis such as susceptibility to disease and interspecies transmission. A central event in TSE disease involves the conversion of the normal host cellular prion protein (PrPC) to a partially protease-resistant, aggregated, disease-associated isoform (PrPSc). TSE-induced pathology is usually associated with PrP-res deposition, but the mechanism of neurodegeneration is not understood. The nature of the infectious agent, called a prion, remains uncertain but is thought to be composed primarily of misfolded PrP, perhaps in complex with another host accessory molecule(s). PrPC is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, and the majority of PrPSc produced in vivo contains this GPI anchor. Membrane association of both normal and disease-associated PrP isoforms may influence many features of prion disease and PrPC function. Our work is focused on elucidating mechanisms of uptake, replication, and spread of prions, in addition to determining the biochemical composition of mammalian prions. In 2009, we have: 1) continued our characterization of how PrPSc is internalized and trafficked in neuronal cells by live cell imaging;2) developed new methods to specifically tag PrPC molecules to visualize their trafficking in uninfected cells and during the course of scrapie infection;3) applied these new methods to reveal new insights into the differential mechanisms of action of anti-TSE compounds and trafficking pathways of PrPSc and PrPC;4) created new compounds for protein labeling that allow analysis by a variety of detection methods;and 5) developed new cell culture models expressing various PrPC mutant proteins. These efforts led to two publications in 2009.